The present invention relates to bacteria counting methods, bacteria counting apparatuses, and bacteria counting reagent kits. More particularly, the present invention relates to bacteria counting methods, bacteria counting apparatuses, and bacteria counting reagent kits for classifying and counting dead bacteria and live bacteria using fluorescent dyes.
Counting the number of bacteria contained in specimens of living organisms or food sources in order to determine whether or not bacteria exist in the food or living organisms is performed in the fields of clinical examinations and food sanitation inspections. Conventional counting methods include culturing, ATP assays, and fluorescent assay methods.
The culturing method is a method of counting bacteria by applying a coating of a specimen onto an agar plate, culturing for 18 to 24 h, and then counting the bacteria in the formed colony. In culturing methods, after the specimen coating is applied to a liquid medium and cultured, growth of the bacteria can be determined by the presence of a suspension.
The ATP assay method is a method of counting bacteria by extracting ATP, which represents bacterial activity, from a specimen, adding a luminous reagent to react with the ATP, and detecting the intensity of the light emitted by the luminous reagent.
The fluorescent assay method stains a specimen using a combination of a fluorescent dye to stain living bacteria and a fluorescent dye to stain dead bacteria, and detects and counts the living bacteria and the dead bacteria via a flow hyetometer and fluorescence microscope.
There are various problems associated with conventional methods, however. For example, the culturing method is basically performed manually, and requires complex operation. Since culturing takes a long time, considerable time must elapse before the bacteria can be counted. Furthermore, only cultural bacteria can be counted.
The ATP assay method requires complex preprocessing, such as the ATP extraction process. Since the bacteria are not counted directly, and the bacteria are counted based on ATP concentration from the intensity of light emission by the luminous reagent, there is a possibility for calculation errors. Furthermore, only bacteria that produce ATP can be counted.
Fluorescent assay requires two types of dyes, which have widely different detection wavelengths for the detection of living bacteria and the detection of dead bacteria, respectively. Accordingly, in order to count living bacteria and dead bacteria, the same sample must be measured using different detection devices for each dye, thereby complicating the structure of the assay device. In addition, the operation of the assay device also becomes complicated.
As described above, the conventional methods are disadvantageous in that they can only count living bacteria, are complicated to operate, or require complex measuring devices capable of counting living bacteria and dead bacteria.
When an antibiotic is added to the specimen containing the bacteria and the change in the number of bacteria is observed over time to determine the antibacterial effect, it is desirable to obtain not only the number of living bacteria, but also the number of dead bacteria. Furthermore, when confirming the presence of bacteria in a specimen beforehand, the bacteria must be counted and a culture study performed if the bacteria are alive, whereas a culture study is unnecessary when the bacteria are dead. Therefore it is desirable to obtain a live bacteria count from the specimen in order to determine whether or not a culture study is required.
European Patent publication No. 1 136 563 A2 describes a method for dying bacteria by applying a cationic surface-active agent to a specimen containing bacteria, then adding a dye. There is no mention of obtaining both a live bacteria count and a dead bacteria count.
In short, a simple method capable of providing both a live bacteria count and a dead bacteria count would be desirable.